Controlling fungi with 5-fluorocytosine



United States Patent 3,368,938 CONTROLLING FUNGI WITH S-FLUOROCYTOSINEJulius Berger, Passaic, and Robert Duschinsky, Essex Fells, N.J.,assignors to Hoffmann-La Roche Inc., Nutley, N.J., a corporation of NewJersey No Drawing. Continuation-impart of application Ser. No. 181,822,Mar. 22, 1962. This application Jan. 13, 1966, Ser. No. 520,371

5 Claims. (Cl. 16758) This application is a continuation-in-part ofUnited States patent application Ser. No. 181,822, filed Mar. 22, 1962(now abandoned), the latter application being a continuation-in-part ofUnited States patent application Ser. No. 176,902, filed Mar. 2, 1962(now abandoned).

This invention relates to the use of S-fluorocytosine in the control offungi. More specifically, this invention relates to the use ofS-finorocytosine in combatting certain fungi which are pathogenic tohumans, animals and plants.

It is known in the art that particular fiuoropyrimidine compounds,including S-fluorocytosine, inhibit the growth of certain bacteria. Ithas now been found that S-fluorocytosine is also quite effective ininhibiting the growth of certain classes of fungi. As used herein, theterm fungi includes both yeasts and molds. The finding thatS-fluorocytosine possesses antifungal activity is quite unexpected.

Thus, by way of example, S-fluorocytosine has been found to be effectiveagainst the organism causing candidiasis. It is particularly effectiveagainst Candida albicans, the species which causes an acute or subacuteinfection in the mouth or throat, skin, nails, vagina, bronchi, lungs orintestinal tract. Additionally, S-fluorocytosine is effective ininhibiting the growth of, for example, aspergilli and penicillia. Moreparticularly, 5-fluorocytosine inhibits the growth of Aspergillus niger,the spoilage agent which causes considerable industrial waste and/ordeterioration in food, leather, textile, paint, paper and alliedindustries. It is similarly effective against the spoilage agentsAspergillus flavus, Pencillz'um digitatum and Penicilliwm citrz'num. Inaddition, S-fluorocytosine is effective in inhibiting the growth ofyeasts, such as, Saccharomyces cerevisiae and Rhodozorula rubra.

The very fact that 5-fluorocyt0sine possesses antifungal properties is,in and of itself, surprising. However, even more remarkable is theextent to which that compound is effective when employed for such use.For example, 5- fluorocytosine has been found to be at least ten timesmore effective in vitro against the organism Candida albicans thanS-fluorouracil. At the same time, however, S-fiuorocytosine is at least20 times less toxic than 5- fluorouracil in the mouse and in the dog.

Thus, in its most comprehensive embodiment, the present inventionresides inthe use of 5-fluorocytosine as an antifungal agent. The use ofS-fluorocytosine-containing preparations to combat systemic Candidaalbicans infections represents a preferred embodiment of the invention.In a more particular embodiment, the present invention providescompositions which contain 5-fluorocytosine as the active antifungalingredient.

The in vitro activity of S-fluorocytosine was tested by the agarcup-plate, diffusion assay procedure which is used in penicillin assay.This procedure is described by Donald C. Grove and Wi liam A. Randall,in the text Assay Methods of Antibiotics, published by MedicalEncyclopedia Incorporated, New York, N.Y., 1955, at pages 2 to 16. Inthe present instance, however, fungi were used as the inoculum ratherthan bacteria. S-fluorocytosine was found to give inhibition zones of 20mm. in diameter, or greater, against Paecilomyces varioti andPenicilliwm digitatum at a concentration of 0.1 mg. per ml. Moreover,

ICC

where a semi-synthetic agar was used in place of the complex nitrogenagar, the sensitivity of yeasts and fungi to 5-fluorocytosine was foundto be extremely great. Thus, at a concentration of 0.01 mg. per ml. ofsolution added to the assay cylinders, zones of 20 to 37 mm. in diameterwere observed against Candida albicans, Saccharomyces cerevisiae andagainst Paecilomyces varioti and Penicillium digitatum. Concentrationsof 1 mg. per ml. of 5- fluorocytosine, in the semi-synthetic agarmedium, produced inhibition zones of 45 to 60 mm. in diameter againstPenicillium citrz'num, Aspergillus flavus and Aspergillus niger. Thetable which follows hereinafter contains a comparison between theantifungal activity of 5-fluorocytosine and the antifungal activity ofthe commercially available antifungal agent which is known under thegeneric name Nystatin. The latter product is a polyene antifungalantibiotic produced by Streptomyces noarsei, Streptomyces aureus andother Streptomyces species.

ANTIFUNGAL SPECTRA IN VITRO OF S-FLUORO- CYTOSINE AND NYSTATIN All ofthe test organisms were grown on a semi-synthet ic vitamin-salts-caseinhydrolysate agar medium, at pH 5.5. The medium was prepared in thefollowing manner: 10 mg. of pyridoxine, 10 mg. of thiamine, 1000 mg. ofinositol, mg. of calcium pantothenate, 0.32 mg. of d-biotin and 100 mg.of niacin were added to a sufiicient quantity of water to provide 1.0liter of a vitamin stock solution. In a separate vessel, 200 grams ofdextrose (anhydrous), 2.2 grams of dibasic potassium phosphate (KH PO4),1.7 grams potassium chloride, 0.5 gram of CaCl -2H O, 0.5 gram MgSO -7-HO, 0.01 gram of FeCl -6H O and 0.01 gram of MnSO -H O were added to asufficient quantity of water to provide 1.0 liter of a sugar and saltsstock solution. A citrate buffer solution was also prepared separatelyby adding 90.0 grams of sodium citrate and 20.0 grams of citric acid toa sufiicient quantity of water to provide 1.0 liter of stock solution.The desired medium was obtained by mixing 25 ml. of the aforementionedvitamin stock solution, 250 ml. of the sugar and salts stock solution,50 ml. of the citrate buffer solution, 40 ml. of a casein hydrolysatestock solution and 15 grams of agar in a sufiicient quantity of water toprovide 1.0 liter of medium. The aforementioned casein hydrolysate stocksolution was obtained by adding 100 grams of Sheffield N-Z amine A to asufiicient quantity of water to provide 1.0 liter of stock solution.Sheiiield N-Z amine A is a casein hydrolysate produced and sold byShefiield Chemical Company, a division of National Dairy ProductsCompany, Norwich, NY. The medium was sterilized by autoclaving for 10minutes.

The cultures which are designated in the table which follows hereinafteras Item Nos. 1, 3 and 4 were grown 18 hours at 35 C. The remainingcultures were grown at 28 C. for sufiicient time to permit good growthof each organism (1-3 days).

[Diameter of inhibition zones in mm.]

MgJml. concentration of- IItIem Test Organism Nystatin fi-fluorocytosineSaccharomyces cerem'st'ae 22 38 29 23 Rhodolorula rubra 37 45 32 20Candida albicans 30 33 27 13 Paeczlomyces vtmo 31 35 27 19 Pemcilliumcztrmum 25 46 32 18 6 Aspergillus flaws 24 52 36 22 7 Aspergillus niger27 49 42 23 8 Pencillz'um digitaium 33 47 27 18 A1; 0.125 rug/ml.

It will be observed from the data set forth in the foregoingtable that-fiuorocytosine is much more etfective than Nystatin, being as much as30 to 500 times as active in various instances. Moreover, it has beenfound that the addition of human blood to a S-fluorocytosine solutiondid not materially decrease the activity of the drug against Candidaalbicans.

As will be seen from Example 1 which follows hereinafter,S-fiuorocytosine is effective, in vivo, against systemic fungalinfections such as Candida albicans infections. Its antifungalproperties, coupled with its low toxicity, render S-fluorocytosineextremely well suited for use in the treatment of fungal infections inhuman, animal and plant hosts. Additionally, the properties ofS-fluorocytosine are such as to render it suitable for use in thetreatment of the spoilage agents which cause industrial waste and/ordeterioration of food.

From a pharmacological standpoint, S-fiuorocytosine has been found to bewell suited for use in treating fungal infections in human hosts. Forexample, S-fiuorocytosine is not toxic; it is not metabolized; and it isquantitatively excreted in the urine. Moreover, S-ftuorocytosine becomesdistributed throughout the mammal organism and it passes the brain-bloodbarrier. Furthermore, sustained blood levels are maintained in humans,half-life of the compound being from 7 to 10 hours.

Clinical trials have demonstrated that S-fiuorocytosine is both safe andetfective in the treatment of fungal infections in humans. Fivepatients,'who had a prior, unsuccessful history of chemotherapy, weretreated for systemic fungal diseases with S-fluorocytosine. The doseschedules varied from 1.0 gram per day, in the case of a child, to 9.0grams per day in the case of an adult. The treatment ranged from 3 to 8weeks. In all instances, the S-fiuorocytosine was administered in theform of 250 mg. tablets, such tablets being given orally in four divideddosages per day. Four of the patients were diagnosed as having asystemic moniliasis infection, the fifth patient having been diagnosedas having cryptococcal meningitis infection. Cryptococcal meningitisinfection is caused by Cryptococcus neoformans. All five patients showedobjective response to S-fiuorocytosine. In the case of the moniliasispatients, response to the treatment with 5- fluorocytosine wasmanifested by cultures of specimens, i.e., blood, urine, bronchialwashings, such cultures being taken before and after therapy. In everyinstance, positive cultures became negative after treatment. In the caseof the cryptococcal meningitis patient, microscopic examination of thecerebrospinal fluid revealed the disappearance of the fungus. Inaddition, clinical signs of improvement were associated with theobjective observations. In all instances, S-fluorocytosine was welltolerated. There was no toxic effect upon bone marrow, thegastrointestinal tract, liver, kidneys or central nervous system.

For pharmaceutical applications, S-fluorocytosine can be used as is orit can be formulated into solutions or suspensions using medicinallyacceptable liquid vehicles such as water or alcohol. Generally, suchsolutions or suspensions will contain at least about 0.1%, by weight, of5-fiuorocytosine. However, under ordinary circumstances, solutions orsuspensions which are more highly concentrated with S-fluorocytosinewill be far more useful. Solutions or suspensions of S-fluorocytosinecan be administered as oral medication. In the alternative, suchsolutions or suspensions can be administered by injection, for example,intravenously, subcutaneously, or intraperitoneally. Local fungalinfections can be treated using, for example, suppositories, ointments,salves, lozenges, etc. which contain S-fiuoroeytosine as the activeingredient. Additionally, 5-fluorocytosine can be formulated intosuitable solid oral dosage forms, such as hard-shell capsules, tablets,etc. In the production of 5-fluorocytosine capsules or tablets, any ofthe adjuvant materials. ordinarily used in formulating such products,can be used. These include, for example, tillers such as coprecipitatedaluminum bydroxide-calcium carbonate, dicalcium phosphate or lactose,disintegrating agents such as maize starch, and lubricating agents suchas talc, calcium stearate, etc. The quantity of S-fluorocytosine whichis incorporated into the various solid dosage forms can be varied withina very wide range. In general, the maximum permissible concentration ofS-ftuorocytosine in any solid dosage form will be determined by purelytechnical considerations. By this it is meant that when producing, forexample, tablets, only such quantities of adjuvant materials need beemployed as are necessary to provide commercially suitable tablets uponcompression of the mixture. Thus, depending on the nature of theadjuvant materials, the maximum amount of S-fiuorocytosine that may beincorporated in each tablet will be subject to rather wide variations.

In the treatment of fungal infections in human hosts, the frequency ofadministration of S-fiuorocytosine-containing compositions will varydepending upon the levels of 5-fluorocytosine present in suchcompositions and the needs and requirements of the patient, as diagnosedby the attending physician. As indicated therefore, dose schedules havebeen varied from 1.0 gram to 9.0 grams per day, the compound having beenadministered orally in the form of 250 mg. tablets. The foregoingnotwithstanding, the dosages mentioned herein are exemplary only andthey are not intended to limit the scope of practice of this invention.It will be readily apparent that, in certain instances, theadministration of quantities of 5- fiuorocytosine substantially lessthan 1.0 gram per day may be desirable whereas, in other instances, theadministration of quantities of S-fluorocytosine in excess of 9.0 gramsper day may be indicated.

When S-fluorocytosine is to be used for food preservative or agriculturepurposes, it can be dissolved or suspended in a suitable solvent and thesolution or suspension, thus obtained, may be sprayed onto food or ontothe plant to be protected. When S-fluorocytosine is to be employed forthe preservation of industrial products, the compound can be added, asis, or suspended or dissolved in a suitable vehicle to the product. Sucha technique could be utilized where the product to be protected is, forexample, a paint. In the treatment of leather, textiles, paper, etc.,S-fluorocytosine could most conveniently be applied thereto in the formof a solution or suspension by spraying or soaking.

For a fuller understanding of the nature and objects of this invention,reference may be had to the following examples which are given merely asfurther illustrations of the invention and are not to be construed in alimiting sense.

Example 1 In this example, aqueous solutions containing varyingconcentrations of S-fiuorocytosine were prepared. Each solution wasobtained by adding S-fiuorocytosine to water and dissolving same thereinby mixing at room temperature. One solution, thus obtained, contained aratio of 1 mg. of S-fluorocytosine for each 1.0 ml. of water. A secondsolution contained a ratio of 2 mg. of S-fluorocytosine for each 1.0 ml.of water. A third solution contained 4 mg. of S-fiuorocytosine for each1.0 ml. of water. A fourth solution contained a ratio of 10 mg. ofS-fiuorocytosine for each 1.0 ml. of water.

These solutions were evaluated for their effectiveness against systemicCandida albicans infections in white mice. The Candida albicansinfections were established in the test animals in the following manner:

The growth from a 24 hour Sabourauds agar slant culture of Candidaalbicans was washed from the surface of the medium in 5 ml. of saline.The suspension was spun in the centrifuge at r.p.m. for 3 minutes tocause the clumps of cells to settle out at the bottom of the tube whilesingle cells remained in the supernatant. The supernatant was decantedand the number of cells per milliliter was determined by hemocytometriccount, using the technique employed for counting leukocytes, asdescribed in the War Department Technical Manual, Methods for LaboratoryTechnicians, Oct. 17, 1941, pages 14 to 18. The suspension was thendiluted to contain 100,000 cells of Candida albicans per ml.

To accomplish the reproducible infection of mice, 0.1 ml. of a 1%suspension of cortisone in saline was injected intramuscularly on themorning and afternoon of the day before infection. On the day ofinfection the animals received 0.1 ml. of the 1% suspension of cortisonein the morning, followed by 0.5 ml. of the 100,000 cells per ml. ofCandida albicans intravenously. On the same afternoon a fourth treatmentof cortisone was administered. Fifth and sixth injections of cortisonewere administered on the morning and afternoon of the day followinginfection.

The treatment with S-fluorocytosine was started on the day of infectionand continued for 21 days. Negative cultures from the kidneys were thecriteria used for the evaluation of antifungal effectiveness of the5-fluor0- cytosine.

Control animals infected intravenously with Candida albicans succumbedbetween the seventh and fourteenth day after infection. Positivecultures were obtained from the kidney on Sabourauds agar.

Table 2, which follows hereinafter, shows the results obtained whenmice, infected intravenously with 50,000 cells of Candida albicans, weretreated intraperitoneally and orally with aqueous solutions ofS-fluorocytosine. -In the case of the 100 mg./kg. dose, two groups oftest animals were administered, in one case orally and in the otherintraperitoneally, 1.0 cc. of a solution containing a ratio of 2 mg. ofS-fluorocytosine in 1.0 ml. of water each day. In the case of theintraperitoneal 50 ing/kg. dose, the test animals were injected each daywith 1.0 cc. of a solution containing a ratio of 1.0 mg. ofS-fiuorocytosine in 1.0 ml. of water. In the case of the oral 200 mg./kg. dose, the test animals received daily, 1.0 cc. of a solutioncontaining a ratio of 4 mg. of 5-fiuorocytosine in 1.0 ml. of water. Inthe case of the oral 500 mg./kg. dose, the test animals received daily1.0 cc. of a solution containing a ratio of mg. of S-fluorocytosine in1.0 ml. of water.

The test results obtained were as follows:

TABLE 2.--THE ACTIVITY OF 5-FLUOROCYTOSINE AGAINST THE OANDIDA ALBICANSINFECTION OF \VHITE MICE Infection: 50,000 cells intravenously in micepreconditioned with cortisone Treatment: 21 days Kidney cultures As willbe seen from the foregoing table, an appreciable effect was observedwhen the test animals were treated with the S-fluorocytosine solutionsby intraperitoneal injection and orally.

Example 2 In this example, S-fluorocytosine was formulated into acompressed tablet in the following manner:

42.8 parts by weight of lactose, 41.8 parts by weight of dicalciumphosphate and 10.0 parts by weight of 5- fluorocytosine were intimatelyadmixed. In a separate vessel, an aqueous paste containing 10.0% byweight of gelatinized starch was prepared. The gelatinized starch usedwas a product which is sold by Corn Products Refining Company, New York,N.Y., under the trade name of Amijel Powder. Thereafter, 3.0 parts byweight of the paste, thus produced, were added to, and mixed with, thelactose-dicalcium phosphate-S-fluorocytosine mixture to form agranulate. The granulate was then comminuted and the finely dividedparticles, thus obtained, were dried at a temperature of from about F.to F. The dried product was subsequently comminuted and thereaftercompressed into tablets using a one-quarter inch flat-faced punch.

Example 3 In this example, tablets containing S-fluorocytosine as theactive ingredient were prepared. The method and the adjuvant mixtureused in producing the tablets of this example were somewhat differentthan the method and adjuvant mixture used in producing the tablets ofExample 2.

In producing the tablets of this example, a mixture was prepared using10.0 parts by weight of S-fiuorocytosine, 88.0 parts by weight oflactose and 2.0 parts by Weight of calcium stearate. The mixture was,thereafter, comminuted and the finely divided powder was compressed intotablets using a one-quarter inch fiat-faced punch.

Example 4 Additional S-fluorocytosine tablets were produced in thefollowing manner: a granulation was first prepared by mixing 27.5 partsby weight of corn starch, 4.5 parts by weight of pre-gelatinizedcornstarch and 68.0 parts by weight of lactose. Thereafter, 88.0 partsby weight of the above-described granulation were mixed with 10.0 partsby weight of S-fluorocytosine and 2.0 parts of calcium stearate. Themixture of subsequently comminuted and compressed into tablets on aone-quarter inch flat-faced punch.

We claim:

1. A method for the treatment of a systemic moniliasis infection in ahuman patient which comprises administering to the infected patient aneffective amount of a composition comprising 5-fluorocytosine and amedicinally acceptable carrier therefor.

2. A method for the treatment of Cryptococcal meningitis infection in ahuman patient which comprises administering to the infected patient aneflective amount of a composition comprising 5-fluorocytosine and amedicinally acceptable carrier therefor.

3. A method for controlling fungi which comprises subjecting the hostfor said fungi selected from the group consisting of animals and plantsto the action of a composition comprising S-fluorocytosine and a carrierthereof.

4. A method of controlling fungi selected from the group consisting ofCandida albicans and Cryptococcus neoformans which comprises subjectinghuman host for said fungi to the action of a composition comprising 5-fluorocytosine and a carrier thereof.

5. The method of claim 4, wherein said fungi is Candida albicans.

References Cited Cecil et al.: A Textbook of Medicine, W. B. SaundersCompany, Philadelphia, Pa. (1955), Pp- 335-336.

ALBERT T. MEYERS, Primary Examiner.

SAM ROSEN, Examiner.

VERA C. CLARKE, Assistant Examiner.

4. A METHOD OF CONTROLLING FUNGI SELECTED FROM THE GROUP CONSISTING OFCANDIDA ALBICANS AND CRYPTOCOCCUS NEOFORMANS WHICH COMPRISES SUBJECTINGHUMAN HOST FOR SAID FUNGI TO THE ACTION OF A COMPOSITION COMPRISING5FLUOROCYTOSINE AND A CARRIER THEREOF.